Anaplasma phagocytophilum (Ap) is an intracellular pathogen causing human granulocytic anaplasmosis (HGA, formerly HGE), an emerging tickborne infection that can be fatal. Ap invades neutrophil granulocytes (PMN), the first cells to infiltrate the tick bite site, via PSGL-1 which normally initiates rolling on inflamed endothelium, leading to extravasation, phagocytosis and death of PMN. But because infected PMN remain in the blood stream and are too short-lived to maintain Ap over extended periods, we hypothesize that other cells may be involved. We found that Ap multiplies in human primary microvascular endothelial cells and lines, causing upregulation of adhesion molecules, adherence of PMN, and increased expression of TNFalpha and CD83 by HL-60 cells. Infected human endothelial cells alter the ratio of MHC-I and II expression, and upregulate galectin-1, which has been found to inhibit PMN transmigration. We hypothesize that Ap infects endothelium, disrupting adhesion-rolling and transmigration to infect PMN, and induces production or display of immunomodulatory substances by infected endothelium. We will examine gene expression with human gene arrays and RNA from endothelial cells, HL-60 and PMN exposed to Ap for various times and conditions, focussing on genes linked to immuneregulation, leukocyte function, and inflammation. We will use confocal microscopy with FISH, antibodies to Ap and cell surface markers to follow Ap from tick to mammal, identify sites of infection, detail endothelial cell invasion and passage to PMN. Our DsRed-expressing tick and endothelial cells will facilitate imaging. Ap will be electroporated with fluorescent protein genes, as already achieved by us with Rickettsiae. Until available Ap are labeled with live cell dyes. Endothelial and PMN receptors will be identified in adhesion assays with antibodies to cell surface markers and lectins. Surface association of Ap and relationship to cellular markers will be studied by FESEM. To identify Ap adhesion proteins, we will screen a cDNA library with blocking antisera, but focus on p100, p130 and MSP2 variants.